The integrins are a family of cell-surface glycoproteins that link the extracellular matrix (ECM) to the cytoskeleton. Integrin-mediated cell adhesion and migration play important roles in a variety of biological processes including embryonic development, wound healing and cancer. The overall objective of the proposal is to identify and characterize integrin interactions with other cytoplasmic proteins, which play essential roles in mediating integrin signaling as well as their affinity regulation. Recent studies from our laboratory has identified a novel interaction between integrin Beta-1 cytoplasmic domain and 14-3-3-Beta which is a member of the 14-3-3 proteins implicated in a variety of cellular functions and signaling pathways. In this application, we propose a series of related experiments to understand the mechanisms by which 14-3-3-Beta interactions with integrin cytoplasmic domain regulate integrin functions in cell spreading and migration. We will generate specific mutants in both integrin Beta-1 and 14-3-3-Beta that are defective in binding to each other. These mutants will then be expressed in cells to examine their potential effects on integrin-mediated cell spreading and migration. We will generate specific 14-3-3-Beta mutants that are defective in its association with other signaling molecules to further evaluate potential roles of association of these molecules with the 14-3-3-Beta/integrin Beta-1 complexes. Lastly, we will examine the effects of 14-3-3-Beta and its mutants on integrin beta-1 clustering and affinity regulation using FRET and soluble ligand binding assays.